Cell cycle progression is a tightly regulated event, the deregulation of which is a hallmark of cancer. Eukaryotic cell cycles are controlled by the temporal association of a cyclin dependent kinase (cdk) with a cyclin partner, leading to the activation of these complexes and the subsequent phosphorylation of critical substrates. The mammalian D-type cyclins and their cdk partners, cdk4 and cdk6, are important regulators of G1 phase progression; little is known about how these cdk/cyclin complexes are regulated, however. This proposal describes the molecular cloning and biochemical characterization of a novel approximate 28 kDa protein (p28) found in cdk6 immunoprecipitates containing D-type cyclins isolated from primary human T- cells in early G1 phase. Curiously, p28 immunoreacts with antibodies generated against p27KIP1, a recently identified cdk inhibitor, yet is distinct from p27KIP1. These findings raise the intriguing possibility that p28 represents a novel cdk inhibitor and that its association with cdk6/ cyclin D complexes has an important regulatory function for G1 phase control. Knowing the cDNA sequence of p28 will allow its expression and association with various cdk/ cyclin complexes as a function of the cell cycle to be analyzed. The possibility that p28 affects the kinase activity of cdk6/ cyclin D complexes or other cdk/ cyclin complexes will be investigated. The minimal domains on p28 required for p28 association with and activity towards cdk/ cyclin complexes will be mapped. Lastly, overexpression of p28 by transient transfection and functional inactivation of the protein by microinjection of anti-p28 antibodies and antisense DNA will be attempted to investigate an in vivo role for p28.